The Effects Of Bio-floc On The Intestinal Bacterial Community Structure Of Grass Carp (Ctenopharyngodon Idellus) And Bighead Carp(Aristichthys Nobilis)

Bio-floc was a kind of reunion material formed by biological flocculation of organisms and inorganic in which the major component were aerobic microorganisms in aquaculture water. The core of the floc were bacterial floc and filamentous bacteria, and then attached by microbial extracellular products (extracellular polymers), intracellular products (poly-β-hydroxybutyrate), polyphosphates, polysaccharides, divalent cation, and so on. Heterotrophic bacteria, denitrifying bacteria, denitrification bacteria, algae, fungi, protozoa and other organisms together constituted the bio-floc. Bio-floc played an important role in the purification of aquaculture water and diets of breeding animals. In this study, bio-floc was used in the process of grass carp and bighead carp breeding. The bacteria structure and diversity of bio-floc during bio-floc formation process and of farming fish intestine were researched by using the PCR-DGGE (PCR-denaturing gradient gel electrophoresis) fingerprinting technique. Combining with the microbial composition, the effects of bio-floc on the intestinal bacterial community structure of grass carp{Ctenopharyngodon idellus) and bighead carp(Aristichthys nobilis) were analyzed.1、Analysis of microbial community structure in different formation stages of bio-flocThe bacterial communities of bio-floc were analyzed by using the PCR-DGGE (PCR-denaturing gradient gel electrophoresis) on the5th,10th, and15th days, respectively. The results indicated that diversity of bacterial species in the Bio-Floc was maximal on the10th day and minimum on the15day. The similarity of bacterial communities was highest on the5th day and10th day, which was67.4%, and was lowest on the5th day and15th day which was40.5%.The acquired sequences of24bands in DGGE gel were performed by BLAST searches against NCBI database, and the sequences including the closely related sequences were aligned with Clustal W program in MEGA4for phylogenetic analysis. The main microbes represented by24main bands in DGGE gel were Alphaproteobacteria, Gammaproteobacteria, Betaproteobacteria, Actinobacteria, Bacilli and Bacteroidetes. Among these bacteria, thea-, β-, and y-proteobacteria are the major bacterial groups, but Actinobacteria bacteria exist on the10th day and the15th day. α-proteobacteria was the dominant bacteria during the entire process. The specific bacteria at different stages were usually the flocculant-producing bacteria:Acidovorax, Aeromonas, Agrobacterium only for5th day; Bacillus, Rhodococcus for10th day and15th day, respectively. The present study demonstrated that the bacterial composition and diversity in the Bio-floc were extremely rich when Bio-floc technology was used in the pond freshwater aquculture systems. From the practical point of view for aquaculture, combining with the important roles of these bacteria in water regulating and food application, Bio-floc technology can be used as an effective new technology for freshwater pond aquaculture system.2、The effects of bio-floc on the intestinal bacterial community structure of grass carp(Ctenopharyngodon idellus)Stocking grass carp to bio-floc (starch as the carbohydrate sources) culture group(TSS level maintain100mg/1)、bio-floc (glucose as the carbohydrate sources) culture group(TSS level maintain100mg/1) and the basal diet (3%of body weight) group. The bio-floc group add2.4%feed of body weight. Bacteria DGGE fingerprinting of intestinal flora of three groups samples were constructed by using PCR-DGGE technique. The analysis result of the DGGE profiles showed that the bacterial community diversity levels were higher in bio-floc groups than in the basal diet group; the similarities between DGGE profiles of bio-floc (starch) and bio-floc (glucose) intestinal flora were approximately50%, but only20%-40%between bio-floc and basal diet group, which indicated that the bio-floc but not the carbohydrate sources style of bio-floc showed apparent effect on the intestinal flora. In sequencing analysis of the acquired DGGE bands, the similarity of the16sequences obtained with sequence in the GenBank was more than95%, and all were attributed to α-、γ-Proteobacteria, Chloroflexi and unclassified bacteria. The sequencing results of specific bands in bio-floc group were showed mainly two types of celluase bactaria Aeromonas and Enterobacter. This suggested that the presence of these bacteria were related to the feeding of bio-floc, the special nutrition composition of bio-floc resulted in the existence of such bacteria.3、The effects of bio-floc on the intestinal bacterial community structure of bighead carp(Aristichthys nobilis)Stocking bighead carp to bio-floc culture group(TSS level maintain100mg/1)、 fertilize culture group[add urea7g(N46%) and superphosphate3g(P2Osl6%), maintain zooplankton level8-10ind/1] and cage hanging water group[cage hang grass carp pond upper water(lm), maintain zooplankton level3-5ind/1]. Bacteria DGGE fingerprinting of intestinal flora of three groups samples were constructed using PCR-DGGE technique. The analysis result of the DGGE profiles showed that the bacterial community diversity level was highest in bio-floc group, followed by fertilize culture group, and was lowest in cage hanging water group fish intestine, which suggested that adding a manually feed improved the intestinal flora species diversity of the bighead carp. The similarities between DGGE profiles of bio-floc group and fertilize culture group were highest, which is approximately50%. The cloning and sequencing results of10characteristic bands showed that:Gluconacetobacter, Chloroflexi, Staphylococcus and unclassified bacteria were the specific intestinal bacteria of bighead carp in bio-floc group; Aeromonas, Clostridium and some unclassified bacteria were the specific intestinal bacteria of the bighead carp in fertilize culture group and the cage hanging water group. The bio-floc reduced the distribution of Aeromonas in bighead carp intestine, thereby effectively limited the disease risk of bighead carp because of the existence of large number of these bacteria. The bacteria related to water quality improvement were detected in the bighead carp of bio-floc group, which reflect that bio-floc play an effective role in the improvement of water quality in aquaculture, and in the special type diet of bighead carp.To sum, the bacteria structure and diversity in different stages of bio-floc formation and the effects of bio-floc on intestinal flora of Ctenopharyngodon idellus and Aristichthys nobilis were researched by using the PCR-DGGE fingerprinting technique, which indicated that bio-floc technique was an effective and advanced tool that can be used in freshwater ponds culture.